In virtually all animal phyla, critical steps in embryonic development are regulated by cell-to-cell, or inductive, signals mediated by secreted growth factors. In particular, members of the transforming growth factor β (TGFβ) superfamily regulate a myriad of cellular and developmental processes, such as mitosis, cell differentiation, embryonic pattern formation and organogenesis. In vertebrate embryos, a variety of TGFβ signals affect germ layer specification, body patterning, cell growth and differentiation (1-4). In embryos of the amphibian Xenopus, distinct TGFβ members induce different cell fates, e.g., activin, Vg1 and nodal induce mesoderm characteristic of the dorsal part of the embryo, such as notochord and muscle. Vg1 and activin also induce endoderm characteristics. In contrast, Bone Morphogenetic Proteins (BMPs) specify mesoderm, such as blood and mesenchyme, and regulate epidermal and neural cell differentiation in the ectoderm (see (5) for a review).
Cells respond to ligands in the TGFβ family by transducing signals directly from cell surface receptor complexes to nuclear DNA targets via the Smad family of proteins (see (4), (6), (7), and (52) for a review).
Smads are related to Drosophila Mad (mothers against decapentaplegic [dpp]) and proteins encoded by three related nematode genes Sma 2, Sma 3, and Sma 4. The terms Sma and Mad have been fused as Smad to unify the nomenclature. There are eight members in the Smad family. Phosphorylated Smads 1, 5 and 8 are functional mediators of BMP family signaling in partnership with Smad 4. Smads 2 and 3 are signal transducers for actions of TGFβ and activins. Smad 6 and Smad 7 function as antagonists to inhibit TGFβ/BMP superfamily signaling. Interestingly, Smad7 is localized in the nucleus and accumulates in the cytoplasm in response to TGFβ signalling (73). Furthermore, expression of both Smad6 and Smad7 is regulated by TGFbs, BMPs, growth factors and cytokines thereby providing for negative feedback regulation of the Smad signalling pathway (53-58). Phosphorylated Smad 1 forms a heteromeric complex with Smad 4 when entering the nucleus and activates transcription of early response genes. The BMP receptors may also signal via the mitogen activated protein kinase. It is likely that BMPs regulate cell cycle progression and thus govern differentiation of mesenchymal stem cells.
Signal transduction in two major pathways, BMP and activin/TGFβ, have been described in detail. Two distinct receptor subunits, the Type I and II transmembrane serine/threonine kinases, form activated complexes upon ligand binding. In these complexes the Type II subunit activates the Type I subunit, which directly phosphorylates and activates particular receptor-regulated R-Smad proteins: BMP receptors target Smad1 and closely-related Smads 5 and 8, while activin and TGFβ receptors target Smad1 and closely-related Smads 2 and 3. Upon activation these R-Smads form a heteromeric complex with Smad4, the “common partner” Smad. This complex translocates to the nucleus, binds to promoters of target genes in cooperation with DNA binding proteins, and activates transcription by recruiting coactivators. A third class of inhibitory Smads (1-Smads), Smad6 and Smad7, function as inhibitors that block Smad-Smad complex formation or Smad-receptor interactions. I-Smads bind to the cytoplasmic domain of receptors or directly to Smad1. Mutations in components at all levels in this pathway are associated with embryonic defects and various cancers, underscoring the importance of this growth factor family in developmental and disease processes. (See, (4), (7) for reviews.) In particular, defects of Smad2 and Smad4 are associated with colon and lung cancer and defects in human Smad4 are associated with pancreatic cancer.
Smads do not have intrinsic enzymatic activity. Thus, the nature of the cellular response to Smad signaling is exquisitely sensitive to the level of Smad protein in the cell. Indeed, alternative cell fate determinations in Xenopus embryos can be achieved by relatively small changes in the amount of Smad protein expressed in the cell (8-11). Therefore, regulating the level of Smad protein in the cell can be used as one means of modulating morphogenetic signaling by the TGFβ superfamily.
Protein modification by covalent attachment of ubiquitin is recognized as a general signal to target proteins for degradation via the proteasome (see (12), and (13) for a review). Targets of selective ubiquitination include transcription factors, cell cycle regulators, signal transduction proteins, and membrane proteins (references in (12)). Selective ubiquitination and degradation of specific target proteins can function as an important mechanism to control cell cycle progression, programmed cell death, differentiation and embryonic development. Dysfunction of the ubiquitination pathway is associated with disease and abnormal development. Ubiquitin ligases are part of a multimeric complex that catalyzes the covalent attachment of ubiquitin, a 12.5 kD polypeptide, to target proteins. Attachment of ubiquitin to its target serves as a molecular “flag” that marks the ubiquitinated protein for proteolytic degradation via an organelle known as the 26S proteosome. There are at least three enzymes involved in conjugating ubiquitin to target proteins, namely, E1, E2 and E3. The E1 enzyme activates a ubiquitin molecule and conjugates it to the E2 enzyme which then either directly attaches ubiquitin onto a target protein, or passes it to the E3 ubiquitin ligase. The E3 recognizes a particular substrate and directs it ubiquitination.
A few examples of developmental regulation by the ubiquitination system have been described in Dictyostelium (14-16) and Drosophila (17-21). Conjugation of ubiquitin to receptors is used in diverse systems to control endocytosis and signalling, as well as receptor steady state levels by both proteasome- and lysosome-mediated degradation (59-60). Direct ubiquitination of membrane receptors has been characterized in a number of systems, although in some cases ubiquitin-dependent regulation does not appear to involve direct conjugation of ubiquitin to the receptor (61-62). Although many cell surface receptors are regulated by ubiquitin-dependent pathways, few E3 ubiquitin ligases that bind to membrane proteins and target them for ubiquitination have been defined. Nedd4, a C2-WW-HECT domain E3 ubiquitin ligase, can regulate the turnover of the amiloride-sensitive sodium channel by binding directly to a PPXY motif present in the carboxy-terminus of the channel (63-66). Furthermore, the RING finger protein, c-cbl, has recently been shown to function as an E3 ubiquitin ligase that binds to the EGF receptor to mediate ubiquitination and downregulation of the receptor complex (67-68). In these examples, ubiquitination of the membrane proteins appears to involve direct interactions between the E3 ligase and the target protein. Whether adaptor proteins might also function to recruit E3 ligases to specific receptor complexes is unknown. The mechanism and targets of ubiquitination in the control of patterning have heretofore remained elusive.
References that are cited throughout the specification by number are listed at the end of the Example. All references cited herein are incorporated by reference.